Compositions and Methods for Augmenting Kidney Function

ABSTRACT

The present invention provides a nutritional food product composed of a prebiotic and an isolated protein for use in reducing elevated levels of nitrogenous waste products in the blood and ameliorating renal failure.

INTRODUCTION

This application is a continuation-in-part of U.S. Ser. No. 12/407,201filed Mar. 19, 2009, which is a continuation of U.S. Ser. No. 10/936,262filed Sep. 8, 2004, which is a continuation-in-part of U.S. Ser. No.PCT/US2002/007554 filed Mar. 13, 2002, each of which are incorporatedherein in their entireties.

BACKGROUND OF THE INVENTION

One of the main functions of the normal, healthy kidney, besides itsregulatory, endocrine, and metabolic functions, is the disposal of wasteproducts. Any impairment of excretory function can lead to theaccumulation of a variety of nitrogenous waste products including, urea,creatinine and uric acid. High concentrations of waste products in theblood stream can exacerbate renal failure and promote kidney stones.Moreover, nitrogenous solutes in the circulating blood promote osmoticdiffusion into the lumen because of the concentration gradient acrossthe intestinal wall. This diffusion mechanism led to the concept of oralsorbents to augment gut-based clearance of nitrogenous waste products.Sorbents or microbes have demonstrated their ability to remove variouscompounds and nitrogenous wastes within the large bowel.

Urea-specific sorbents such as synthetic polymers and modifiedpolysaccharides have been evaluated for the removal of urea and othernitrogenous wastes via the gut. Other sorbents such as oxidized starch,activated charcoal, and carob flour have also been investigated for thein vivo elimination of uremic toxins with some success. Prakash & Chang((1996) Nature Medicine 2:883-88) demonstrated that microencapsulated,genetically-engineered E. coli DH5 .are effective in removing urea andammonia in an in vitro system. The same researchers obtained similarresults in oral administration of E. coli DH5 cells in a uremic ratanimal model. Bliss et al. ((1996) Am. J. Clin. Nutr. 63:392-398) havedemonstrated that supplemental gum arabic fiber increases fecal nitrogenexcretion and lowers urea nitrogen concentration in chronic renalfailure patients consuming a low protein diet. Reinhart et al. ((1998)Rec. Adv. In Canine and Feline Nutr. Iams Nutrition SymposiumProceedings. Vol. II:395-404) found that canine renal patients fed adiet containing a fermentable fiber blend improved clinical end-stagerenal disease status, suggesting that specific nutritional alterationallows repartitioning of nitrogen excretion away from the kidney andinto the feces by colonic fermentation or additional bacterial growth.

Prebiotic components are food ingredients that enhance the actions ofprobiotic components in the digestive tract. In this synergistic orrelationship a probiotic component, such as Bifidobacteria, metabolizesundigested carbohydrates, such as dietary fibers, oligosaccharides,etc., to produce short-chain fatty acids such as acetate, propionate andbutyrate. These short-chain fatty acids may promote intestinal cellgrowth, enhance water and mineral absorption, and prevent yeast, mold,and pathogenic bacterial growth. In addition, probiotic components mayantagonize pathogens directly through production of antimicrobial andantibacterial compounds such as cytokines and butyric acid (De Vuyst andVandamme. Antimicrobial potential of lactic acid bacteria. In: De VuystL, Vandamme E L, eds. Bacteriocins of lactic acid bacteria. Glasgow,United Kingdom: Blackie Academic and Professional; 1994:91-142; Dodd andGasson. Bacteriocins of lactic acid bacteria. In: Gasson M J, de Vos WM, eds. Genetics and biotechnology of lactic acid bacteria. Glasgow,United Kingdom: Blackie Academic and Professional; 1994:211-51;Kailasapathy and Chin (2000) Immunol. Cell. Biol. 78(1):80-8), reducegut pH by stimulating the lactic acid-producing microflora (Langhendrieset al. (1995) J. Pediatr. Gastroenterol. Nutr. 21:177-81), compete forbinding and receptor sites that pathogens occupy (Kailasapathy and Chin(2000) Immunol. Cell. Biol. 78(1):80-8; Fujiwara et al., (1997) Appl.Environ. Microbiol. 63:506-12), improve immune function and stimulateimmunomodulatory cells (Isolauri et al. (1991) Pediatrics 88:90-97;Isolauri et al. (1995) Vaccine 13:310-312; Rolfe (2000) J. Nufr.130(2S):396S-402S), compete with pathogens for available nutrients andother growth factors (Rolfe (2000) J. Nufr. 130(2S):396S-402S), orproduce lactase which aids in lactose digestion.

U.S. Pat. No. 4,022,883 discloses a method for alleviating uremicsymptoms in persons suffering from renal failure comprisingadministering orally thereto an effective dosage of a cell mass of anon-pathogenic soil bacteria selected from the group consisting of anurea degrading bacterium, a creatine degrading bacterium, a creatininedegrading bacterium and an uric acid degrading bacterium wherein theurea degrading bacterium is a species of Serratia; the creatininedegrading bacterium is a non-fluorescent Pseudomonas, Rhizobium,Agrobacterium, Corynebacterium ureafaciens, Arthrobacter ureafaciens, E.coli, or Pseudomonas aeruginosa; and a uric acid degrading bacterium isBacillus subtilis, a non-fluorescent Pseudomonas, Bacillus fastidosus,Micrococcus dentrificans, Mycobacterium phlei, Aerobacter aerogenes.

U.S. Pat. No. 4,218,541 teaches a method for converting urea to inocuousproducts. The method involves obtaining a culture of at least onemicroorganism selected from the group of Enterobacter agglomerans, GroupD Streptococcus, Bacilli, and Pseudomonad or mixtures of saidmicroorganisms and adding the culture to a composition containing urea.

U.S. Pat. No. 4,970,153 discloses a method of producing urease and moreparticularly a method of producing acid urease by the cultivation ofLactobacillus fermentum TK 1214. This patent further teaches the use ofsuch acid urease or the decomposition of urea contained in fermentationfood products.

U.S. Pat. No. 5,116,737 teaches a method for growing acid-producingbacterial cultures, such as diary cultures, wherein the culture isselected to contain a urease-producing strain of bacteria and the mediumused for the culturing contains added urea. Urease-producing strains ofStreptococcus thermophilus and Bifidobacterium are also disclosed.

U.S. Pat. No. 5,716,615 discloses a pharmaceutical compositioncontaining several different bacteria including Streptococcusthermophilus, Lactobacilli and Bifidobacteria wherein the bacteria arepresent in the composition at a total concentration of 1×10¹¹ to 1×10¹³per gram. An excipient consisting of maltodextrin, microcrystallinecellulose, maize starch, levulose, lactose or dextrose is furthertaught. Methods of using the pharmaceutical composition are alsodisclosed which include treatment of a gastrointestinal disorder andhypercholesteremia or modulating a host's immune response.

SUMMARY OF THE INVENTION

The present invention is a protein-rich nutritional product composed ofat least one prebiotic and at least one isolated edible protein. In oneembodiment, the probiotic component is selected from the group of aLactobacillus, Bacillus, Streptococcus Bifidobacteria, Saccharomyces orLeuconostoc. In other embodiment, the at least one isolated protein isselected from the group of whey proteins or isolates, concentrates orhydrolysates thereof; milk proteins or isolates, concentrates orhydrolysates thereof whey growth factor extract; glutamine peptide; eggalbumen; soy proteins or isolates, concentrates or hydrolysates; orcaseinates.

This nutritional composition can be in the form of a food product,dietary supplement, or medical food which, upon ingestion, will promotea healthy intestinal microenvironment, provide a source of protein, andassist in the elimination of nitrogenous waste products that can buildup in concentration in the circulating blood. Increased concentrationsof the wastes are known to exert a negative impact on an individual'sphysiology and contribute to a decreased sense of well-being and generalmalaise. Methods for removing nitrogenous waste products from the bloodand ameliorating renal failure using the nutritional product of theinvention are also provided.

DETAILED DESCRIPTION OF THE INVENTION

Nitrogenous waste products accumulating in the blood stream havedetrimental affects on health. Removal of nitrogenous wastes bydiverting them into the colon is a viable approach to decrease thenegative impact that waste product accumulation has on an individual'sphysiology. The present invention combines the properties of a probioticand edible protein into a product to both provide a source of proteinand effectively reduce the blood concentration of nitrogenous wasteproducts.

A probiotic component of the present invention refers to a mono or mixedculture of live or freeze-dried microorganisms which, when applied toman or animal, beneficially affects the host by improving the propertiesof the indigenous microflora, such as bifidobacterium organisms thatmetabolize undigested carbohydrates and are beneficial to an individual.Probiotic components of the present invention are selected for theirability to exert a beneficial effect on the host, survive transitthrough the intestinal tract, to adhere to intestinal epithelial celllining, to produce of anti-microbial substances towards pathogens and/orto stabilize the intestinal microflora. Furthermore, a probioticcomponent should have a good shelf-life. Products of the presentinvention generally contain a large number of viable cells at the timeof consumption, and are non-pathogenic and nontoxic. Examples ofprobiotic components include, but are not limited to, Bifidobacteriumspp. (e.g., bifidum, longum, infantis), Lactobacillus spp. (e.g.,bulgaricus, acidophilus, lactis, helveticus, casei, plantarum, reuteri,delbrueckii, chamnosus, johnsonii, paracasei), Streptococcus spp. (e.g.,thermophilus, diacetilactis, cremoris, durans, faecalis), Saccharomycesspp. (e.g., pombe, boulardii), Leuconostoc spp. (e.g., citrovorum,dextranicum) and Bacillus sp. (e.g., pasteurii). In one embodiment, theprobiotic component is composed of at least one, at least two, or atleast three microorganisms from the genera Bifidobacterium,Lactobacillus, Streptococcus, Saccharomyces, Leuconostoc and Bacillus.In another embodiment, the probiotic component is composed of at leastone microorganism from the genera Bifidobacterium, Streptococcus orLactobacillus. In a further embodiment, the probiotic component iscomposed of at least two microorganisms from the genera Bifidobacterium,Streptococcus or Lactobacillus. In a particular embodiment, theprobiotic component is composed of Bifidobacterium longum, Streptococcusthermophilus and Lactobacillus acidophilus.

Microorganisms also useful in the invention are those that have theability, either through natural selection or by genetic manipulation, tocatabolize various nitrogenous compounds (e.g., urea, creatinine, uricacid and ammonia) by expressing or overexpressing one or more cognatecatabolic enzymes. Exemplary microorganisms are those having an elevatedlevel of urease or creatininase secretion.

A microorganism exhibiting elevated levels of catabolic enzyme secretioncan be selected or trained by exposing a selected microorganism onincreasing amounts of the metabolite of interest (e.g., urea,creatinine, uric acid and ammonia). For example, it has been found thata standard strain of Streptococcus thermophilus can be trained toexpress elevated levels of urease by sequential passage of the strain onincreasing amounts of urea, e.g., a single colony growing on 0.5% ureais selected and applied to medium containing 1.0% urea, a single colonygrowing on 1.0% urea is selected and applied to medium containing 2.0%urea, etc. Using such a method, a S. thermophilus strain having theability to grow on 5% urea was isolated. This strain proliferated inartificial intestinal fluid (AIF, US Pharmacopeia) in the pH range of5.5 to 7.5, characteristic of the colon environment; used urea as a solenitrogen source; and catabolized urea in the presence of other nitrogensources. It was found that urea hydrolysis was growth- and pH-dependentand that urea concentrations could be reduced by this strain from 300mg/dL to 20 mg/dL within 24 hours at pH 6.3 when inoculated in AIF at aninitial density of 10⁹ cfu/mL. Moreover, this strain survived 3 hours inacidic pH 3.0 with only a one-log loss in cfu and was able to passthrough bile. In addition, this strain did not appear to exhibit anyresistance to eight commonly used antibiotics. Therefore, these dataindicate that a specifically selected or trained bacterial isolate canbe used as a urea-targeted component in a product of the presentinvention.

Elevated levels of secretion can also be obtained by overexpressing thegene of interest (e.g., via multiple copies or a promoter driving highlevels of expression) in a prokaryotic microorganism of interest such asBifidobacterium, Lactobacillus, Streptococcus, Leuconostoc or Bacillus,or a eukaryotic microorganism such as Saccharomyces. The gene ofinterest can be under the regulatory control of an inducible orconstitutive promoter. Promoters for use in recombinant prokaryoticexpression vectors are well-established in the art and can include thebeta-lactamase (penicillinase) and lactose promoter systems (Chang etal. (1978) Nature 275:615; Goeddel et al. (1979), Nature 281:544), atryptophan (trp) promoter system (Goeddel et al. (1980) Nucleic AcidsRes. 8:4057; EPO App. Publ. No. 36,776) and the tac promoter (De Boer etal. (1983) Proc. Natl. Acad. Sci. USA 80:21). While these are commonlyused promoters which are commercially available, one of skill in the artcan appreciate that any other suitable microbial promoter can be used aswell. Nucleic acid sequences encoding suitable prokaryotic promotershave been published thereby enabling one of skill in the art to readilyisolate these promoters (e.g., by standard cloning or PCR methodologies)for cloning into plasmid or viral vectors (Siebenlist et al. (1980) Cell20:269). The promoter and Shine-Dalgarno sequence (for prokaryotic hostexpression) are operably-linked to the DNA encoding the gene ofinterest, i.e., they are positioned so as to promote transcription ofthe messenger RNA from the DNA, and subsequently introduced into asuitable host cell.

Eukaryotic microbes such as yeast cultures can also be transformed withsuitable protein-encoding vectors. See e.g., U.S. Pat. No. 4,745,057.Saccharomyces cerevisiae is the most commonly used among lowereukaryotic host microorganisms, although a number of other strains arecommonly available. Yeast vectors can contain an origin of replicationfrom the 2 micron yeast plasmid or an autonomously replicating sequence(ARS), a promoter, DNA encoding the desired protein, sequences forpolyadenylation and transcription termination, and a gene encoding for aselectable marker. An exemplary plasmid is YRp7, (Stinchcomb et al.(1979) Nature 282:39; Kingsman et al. (1979) Gene 7:141; Tschemper etal. (1980) Gene 10:157). This plasmid contains the trp1 gene, whichprovides a selection marker for a mutant strain of yeast lacking theability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1(Jones (1977) Genetics 85:12). The presence of the trp1 lesion in theyeast host cell genome then provides an effective environment fordetecting transformation by growth in the absence of tryptophan.

Suitable promoting sequences in yeast vectors include the promoters formetallothionein, 3-phospho-glycerate kinase (Hitzeman et al. (1980) J.Biol. Chem. 255:2073) or other glycolytic enzymes (Hess et al. (1968) J.Adv. Enzyme Reg. 7:149; Holland et al. (1978) Biochemistry 17:4900),such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase,pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphateisomerase, phosphoglucose isomerase, and glucokinase. Suitable vectorsand promoters for use in yeast expression are commercially available andfurther described in Hitzeman et al., EP 73,657.

As will be understood by those of skill in the art, expression vectorscontaining polynucleotides which encode a degradative enzyme ofinterest, e.g., a urease or creatininase, can be designed to containsignal sequences which direct secretion of enzyme of interest through aprokaryotic or eukaryotic cell membrane. Such signal sequences arewell-established in the art and can be taken from other enzymes/proteinsknown to be secreted into the extracellular environment.

Transforming the microorganisms as defined herein, describes a processby which exogenous DNA is introduced into and changes a recipient cell.It can occur under natural or artificial conditions using variousmethods well-known in the art. Transformation can rely on any knownmethod for the insertion of foreign nucleic acid sequences into aprokaryotic or eukaryotic host cell. The method is selected based on thetype of host cell being transformed and can include, but is not limitedto, viral infection, electroporation, heat shock, lipofection, andparticle bombardment. Such “transformed” cells includestably-transformed cells in which the inserted DNA is capable ofreplication either as an autonomously replicating plasmid or as part ofthe host chromosome. This also includes cells which transiently expressthe inserted DNA or RNA for limited periods of time.

As will be appreciated by the skilled artisan, a microorganism can alsobe exposed to a mutagen to cause changes in the genetic structure of theorganism so that it expresses elevated levels of a catabolic enzyme ofinterest.

Transformed or mutagenized strains are subsequently selected for theability to grow in the presence of the metabolite which is degraded bythe catabolic enzyme of interest. By way of example, a straintransformed with nucleic acid sequences encoding a urease is selectedfor high levels of urease secretion by growing said strain on highlevels of urea. Levels of urease secretion can also be detected usingstandard enzymatic assays. As disclosed herein, the strain can besequentially subcultured on increasing levels of urea to further enhanceurease secretion. One embodiment of the present invention provides aurease-secreting strain of Bacillus pasteurii, Streptococcusthermophilus or Saccharomyces pombe. In another embodiment, aurease-secreting strain is at least one of the microorganisms of aprobiotic component composed of at least two or at least threemicroorganisms. In a further embodiment, a urease-secreting strain is atleast two of the microorganisms of a probiotic component composed of atleast three microorganisms.

The probiotics according to the invention can be obtained byfermentation and can be stored after fermentation and before addition tothe composition of the present invention for a time and at a temperaturethat prevents substantial loss of probiotic cfu. For example, theprobiotic component can be fermented until a final concentration of 10⁶to 5×10¹⁰, or 10⁷ to 10¹⁰, or 10⁸ to 10⁹ cfu per mL of fermented mediumis achieved.

When the probiotic component is a mono culture, said mono culture is100% of the probiotic component. When the probiotic component iscomposed of at least two microorganisms, each microorganism can be 10,15, 20, 30, 40, 50, 60, 70, 80, or 90% of the probiotic component,wherein the total of all microorganisms is 100%. An exemplary probioticcomponent is composed of about 10-15% L. acidophilus, about 10-15% B.longum and about 70-80% S. thermophilus (e.g., a ratio of approximately1:1:8, respectively).

The probiotic component or urease-secreting microorganism is included ata concentration of 10⁸ cfu/mL, 10⁹ cfu/mL, 10¹⁰ cfu/mL, 10¹¹ cfu/mL, or10¹² cfu/mL when added as a liquid or 10⁸ cfu/g, 10⁹ cfu/g, 10¹⁰ cfu/g,10¹¹ cfu/g, or 10¹² cfu/g when added as a freeze-dried powder. In oneembodiment, the probiotic component is about 20% to about 70% of thetotal product weight, In particular embodiments, the probiotic componentis about 50% of the total product weight.

Edible nutritional proteins and their derivatives provide specificbenefits, in particular in subjects with stage 5 renal failure whoexhibit significant protein loss. For example, whey protein isolate(WPI) and milk protein isolate (MPI) have been shown to effectivelyprovide a gain in lean muscle mass. WPI is high in branched-chain aminoacids. MPI is primarily casein, shown effective in promoting musclegrowth. Egg protein (albumen) also is high in amino acid content. Wheyprotein hydrosylate (WPH) has been linked to improved nitrogen retentionand growth in rats. Accordingly, in particular embodiments, the edibleprotein of the invention is a whey protein or isolate, concentrate orhydrolysate thereof; milk protein or isolate, concentrate or hydrolysatethereof; whey growth factor extract; glutamine peptide; egg albumen; soyprotein or isolate, concentrate or hydrolysate; or caseinate. In theinstant composition, the protein is added as an ingredient per se and isnot sourced from other ingredients such as peanut pieces. In thisrespect, the protein is isolated, i.e., obtained from its natural sourceas an extract or purified protein (e.g., greater than 90% homogenous tothe protein with fats and carbohydrates removed).

When the protein is a single edible protein, said protein is 100% of theprotein component. When the protein component is composed of two or moreprotein, each protein can be 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, or90% of the protein component, wherein the protein component total is100%.

The amount of protein component added to the composition of theinvention is provided in the range of 10 to 50 grams per serving. In oneembodiment, the protein component is not less than 10 grams and not morethan 100 grams per serving. In one embodiment, the protein component isabout 20% to about 70% of the total product weight, In particularembodiments, the protein component is about 50% of the total productweight. When the product further includes addition additives, thepercent of the protein can be decreased to accommodate the additionaladditive.

In some embodiments, the composition of the invention further includes aprebiotic component. A prebiotic refers to a non-digestive food thatbeneficially affects the host by selectively stimulating the growthand/or activity of one or more non-pathogenic bacteria in the colon.Prebiotic components of the present invention are considered to haveanti-carcinogenic, anti-microbial, hypolipidemic and glucose modulatoryactivities. They can also improve mineral absorption and balance.Furthermore, bacteria belonging to the Bifidobacterium and Lactobacillusfamilies are stimulated by the presence of the prebiotic component andproliferate. Pharmacokinetically, the prebiotic components reach thecolon largely intact. An exemplary prebiotic component includes, but isnot limited to, an oligosaccharide such as fructo-oligosaccharide orinulin, isomaltose oligosaccharide, trans-galacto-oligosaccharide,xylo-oligosaccharide, or soy-oligosaccharide; a pyrodextrin such asarabinogalactan, lactilol, lactosucrose, or lactulose; or a fiber sourcesuch as oat gum, pea fiber, apple fiber, pectin, guar gum, psylliumhusks, glucomannan or guar gum hydrolysate (BENEFIBER, NovartisPharmaceuticals). In one embodiment, the prebiotic component is composedof at least one, at least two, or at least three non-digestive foods(e.g., oligosaccharides, pyrodextrins or a fiber source). In anotherembodiment, an oligosaccharide is at least one of the non-digestivefoods of a prebiotic component composed of at least two or at leastthree non-digestive foods. In yet another embodiment, a fiber source isat least one of the non-digestive foods of a prebiotic componentcomposed of at least two or at least three non-digestive foods. In afurther embodiment, a fiber source is at least two of the non-digestivefoods of a prebiotic component composed of at least three non-digestivefoods. In a still further embodiment, the prebiotic component iscomposed of at least one of the following non-digestive foods oflactulose, psyllium husks and guar gum hydrolysate. In particularembodiments, the prebiotic component is composed of lactulose, psylliumhusks and guar gum hydrolysate.

A nutritional product combining the beneficial properties of a probioticand edible food protein can include a food product, dietary supplement,comestible medical food or pharmaceutical product. The ingestion of saidproduct provides both a protein source and reduces the bloodconcentration of nitrogenous waste products that accumulate in thecirculating blood stream. These waste products of the present inventioncan be of an endogenous origin such as normal or abnormal metabolicroutes or bacterial putrefaction. Furthermore, the waste products can beof an exogenous origin as in dietary intake of proteins and amino acids.Furthermore, repeated ingestion of the product will have a highlybeneficial effect upon the intestinal microflora by localization andcolonization in the large intestine of microbes known to promote ahealthy intestinal microenvironment.

A product of the present invention can take the form of a food productincluding, but is not limited to, a health bar, health drink, yogurt,dahi, ice cream, frozen yogurt or other frozen food product. In additionto containing protein and probiotic components, the product of thepresent invention can further containing various fillers or additives.

Optional additives of the present composition include, withoutlimitation, pharmaceutical excipients such as magnesium stearate, talc,starch, sugars, fats, antioxidants, amino acids, proteins, nucleicacids, electrolytes, vitamins, derivatives thereof or combinationsthereof. In one embodiment, an additive of the product is carob flour,for example, locust bean gum. In another embodiment, an additive is amushroom extract from Agaricus bisporus. In particular embodiments, agel cap contains fillers such as magnesium stearate, talc and starch.

Further, to increase the palatability of a food product containing aprotein and probiotic, it may be desirable to add flavors, sweeteningagents, binders or bulking agents.

Flavors which can optionally be added to the present compositions arethose well-known in the art. Examples include, but are not limited to,synthetic flavor oils, and/or oils from plants leaves, flowers, fruitsand so forth, and combinations thereof are useful. Examples of flavoroils include, but are not limited to, spearmint oil, peppermint oil,cinnamon oil, and oil of wintergreen (methylsalicylate). Also useful areartificial, natural or synthetic fruit flavors such as citrus oilsincluding lemon, orange, grape, lime, and grapefruit, and fruit essencesincluding apple, strawberry, cherry, pineapple and so forth.

Sweetening agents can be selected from a wide range of materials such aswater-soluble sweetening agents, water-soluble artificial sweeteners,and dipeptide-based sweeteners, including salts thereof and mixturesthereof, without limitation.

Binders can be selected from a wide range of materials such ashydroxypropylmethylcellulose, ethylcellulose, or other suitablecellulose derivatives, povidone, acrylic and methacrylic acidco-polymers, pharmaceutical glaze, gums (e.g., gum tragacanth), milkderivatives (e.g., whey), starches (e.g., corn starch) or gelatin, andderivatives, as well as other conventional binders well-known to personsskilled in the art. Examples of bulking substances include, but are notlimited to, sugar, lactose, gelatin, starch, and silicon dioxide.

When the above-mentioned additives are included in the product of thepresent invention, they are generally less than 15% of the total productweight. In particular embodiments, they are less than 5 to 10% of thetotal product weight.

Depending on whether the product is to be consumed by an adult human,child or animal (e.g., companion animal or livestock), it can beproduced in various sizes and with various ingredients suitable for theintended recipient. Further, because the probiotic and proteincomponents of the present invention are generally recognized as safe,they can be consumed one, two or three times daily or more.

The present invention also relates to a method for removing nitrogenouswaste products from the blood of an individual with elevated levels ofnitrogen-containing waste products. The method involves administering aneffective amount of a product of the present invention so that thelevels of nitrogenous waste products in the blood are decreased orreduced, desirably to a normal range. For example, normal levels ofcreatinine in the blood are in the range of 0.6-1.2 mg/dL, whereasnormal blood urea nitrogen (BUN) levels range from 7-18 mg/dL and normaluric acid levels in males and females is in the range of 2.1 to 8.5mg/dL and 2.0 to 7.0 mg/dL, respectively. Further, a BUN/creatinineratio of 5-35 is indicative of normal levels of nitrogenous wasteproducts in the blood. As one of skill in the art can appreciate, meansfor determining the levels of nitrogenous wastes are well-known to theskilled laboratory clinician.

As products of the present invention can reduce the levels ofnitrogenous waste products in the blood of a mammal with chronic renalfailure, these compositions are useful in a method for amelioratingrenal failure. The method involves administering a product of thepresent invention to a subject having or at risk of having renalfailure. Subjects having or at risk of having renal failure includethose with diabetic nephropathy, hypertensive nephrosclerosis,glomerulonephritis, interstitial nephritis, or polycystic kidney diseasewherein nephron function is impaired thereby decreasing glomerularfiltration rate. Desirably, an effective amount of a product forameliorating renal failure is an amount sufficient to effect beneficialor desired results, including clinical results, and, as such, aneffective amount of a product is one which results in the alleviation oramelioration of one or more symptoms associated with renal failure(e.g., a build up of uremic solutes), diminishment of extent of disease,stabilized (i.e., not worsening) state of disease by supporting healthybowel function, delay or slowing of disease progression, or ameliorationor palliation of the disease state. Amelioration can also meanprolonging survival as compared to expected survival if not receivingtreatment. In particular embodiments, the instant composition isadministered to a subject with renal failure and a protein deficiency.

It is further contemplated that compositions of the present inventioncontaining proteins and probiotics may have further utility in theproviding energy and overall health and well-being in subjectsundergoing cancer therapy.

EXAMPLE 1 Yogurt Food Product

Yogurt or frozen yogurt can be prepared from one gallon of commerciallyavailable whole, homogenized, pasteurized milk which is heated toboiling and quickly allowed to cool to approximately 45° C. To this isadded approximately one ounce of yogurt starter culture containinglactic acid bacteria of the genus Lactobacillus, Streptococcus, Bacillusor Bifidobacteria. The mixture is mixed well and allowed to ferment at37° C. for 10 to 12 hours. One or more proteins, prebiotics, whole fruitadditives, flavoring, sweetening agents, binders, or other additives canbe combined and added to the yogurt to obtain a product of desiredconsistency or to suit the palette of the prospective consumer. In oneembodiment of the present invention, a food product comprises componentsto meet the special dietary needs of individuals with renalinsufficiency.

EXAMPLE 2 Health Bar

Health bars can be prepared by combining various excipients, such asbinders, additives, flavorings, colorants and the like, along with theprobiotic (e.g., Lactobacillus, Streptococcus, Bacillus orBifidobacteria) and protein such as soy protein isolate, and mixing to aplastic mass consistency. The mass is then either extruded or molded toform “candy bar” shapes that are then dried or allowed to solidify toform the final product.

EXAMPLE 3 Medical Food

A medical food can be prepared by combining rolled oats, dehydratedapples, honey, inulin, carob flour, cinnamon, sugar, vanilla extract,and protein such as whey protein isolate, and lyophilized cultures of L.acidophilus and or L. fermentum, a Bifidobacteria, and Streptococcusthermophiles (10⁸-10¹⁰ cfu each). These ingredients are mixed inappropriate proportions with a prebiotic and formed into a rectangularbar approximately 12.5 to 15 centimeters in length, 3 to 4 centimetersin width and 1 centimeter in height and placed into a sterile vacuumoven for 12 to 24 hours to obtain an edible food product of the desiredconsistency.

EXAMPLE 4 Minipig Model of Chronic Uremia

The effects of a composition of the present invention in the form of agel cap product or formulation admixed with the pig chow were testedusing an established model of chronic uremia (mild to moderate) in theGottingen strain of miniature swine (Willis, et al. (1997) J. Endourol.11(1):27-32). At sexual maturity (3 months of age) these pigs weigh 8-11kg, and are about half the size of all other strains of minipigs. Thepigs grow slowly and double their body weight in about 6 months.

In this uremic model, ⅚ of the renal mass is removed through bilateralflank incisions (McKenna, et al. (1992) J. Urol. 148(2 Pt 2):756-9). Onekidney is entirely removed, and both poles of the contra lateral kidneyare removed with the aid of electro-cautery (for scoring the renalcapsule and about 2 mm of parenchyma), surgical staples, and Gel foamsutured over the exposed parenchyma of the remnant. The contra lateralkidney is removed during the same surgical procedure by gross dissectionand ligation and section of the renal artery and vein and ureter. Thepigs used in this analysis characteristically experienced a large, acuteelevation of creatinine and Blood Urea Nitrogen (BUN) concentrations inplasma; followed by a decline over 1-2 weeks to values that stabilizedwell above baseline. These pigs maintained their appetite and stableuremia for 3-6 months, maintained or gained body weight, and behaved nodifferently than normal control pigs. Hematocrits declined slowly as theuremia progressed and hemoglobin concentrations also declined over time.After the surgery, the pigs were allowed to recover for 3-4 weeks priorto administering the compositions.

Using this model, several different blinded formulations in the form ofgel caps or admixed with the pig chow were tested as gut-based therapyfor uremia. Formulations administered with the pig chow did notdemonstrate any significant differences either in BUN or creatininevalues. However, ⅚^(th) nephrectomized mini pigs (n=6) given aformulation containing four microbial strains (L. acidophilus, L.bulgaricus, B. longum and S. thermophilus; 10×10⁹ CFU/gel cap) exhibitedcontinued body weight gains of approximately 31% and decreased BUN andcreatinine levels of approximately 13% each. Similarly, two ⅚thnephrectomized mini pigs given a formulation containing three microbialstrains (L .acidophilus, B. longum and S. thermophilus; 10×10¹⁰ CFU/gelcap) also showed a decrease in BUN (average 21%) and creatinine (average29%) levels although the body weight of one mini pig increased (6%) andthe other decreased (20%). Additional formulations were tested in theform of gel caps or admixed with food and in general the resultsindicated that oral treatment with a probiotic formulation effectivelyand significantly reduced plasma creatinine concentrations by 22.5±8.5%in a stable porcine model of chronic renal failure. Small sample sizemay have prevented detection of corresponding reductions in BUN andelevation of hematocrit.

EXAMPLE 5 Animal Dosing

Table 1 provides a suitable approximate serving dosage of a nutritionalproduct for administration to an animal such as a companion animal.

TABLE 1 Weight pounds (Kg) Morning Dose Evening Dose Less than 2.2 lbs 10  (<1 Kg) 2.2-4.4 lbs 1 1 (1-2 Kg) 4.4-8.8 lbs 2 1 (2-4 Kg)  8.8-17.6lbs 2 1-2 (4-8 Kg) 17.6-35.2 lbs 2 2  (8-16 Kg) 35.2-70.4 lbs 2-3 2-3(16-32 Kg) More than 70.4 lbs 3 3 (>32 Kg)

EXAMPLE 6

Urease-Secreting Strains of Streptococcus thermophilus

This example discloses the isolation and selection of a high levelurease-secreting strain of Streptococcus thermophilus. Three isolates ofgram-positive, lactic acid-producing non-pathogenic cocci ofStreptococcus thermophilus were isolated from various sources anddesignated KB4, KB19, and KB25. KB4 was isolated from a probioticproduct, KB19 was isolated from a commercial yogurt product and KB25from Dahi yogurt (from India).

Growth rates and urea hydrolysis of these bacteria in the intestinal pHrange (pH 5.5, 6.3 and 7.5) were determined by transferringexponentially growing cultures of KB19, KB4 and KB25 into modifiedArtificial Intestinal Fluid M2 (AIF, US Pharmacopeia) supplemented with100 mg/dL filter-sterilized urea, 100 μM NiCl₂, 10% MRS broth, dextroseto final concentration of 1%, and 0.3% yeast extract, wherein theinitial cell density was 10⁹ cfu/mL. Pancreatin was omitted from therecipe to allow the evaluation of bacterial growth by direct OD600 nmmeasurement. Urea concentration in the supernatants (% of control) andgrowth (OD600 nm) were measured every 4 hours.

Concentration of urea in the supernatants of bacterial cultures wasmeasured using the protocol and standards supplied with the Blood UreaNitrogen Reagent Kit (535, SIGMA, St. Louis, Mo.). Urea hydrolysis wasmonitored by comparing urea-nitrogen concentrations in bacterialsupernatants to appropriate control medium incubated in the sameconditions and expressed as percent of control. Four to nine independentexperiments were conducted and Student t-test was used for statisticalanalysis.

Under similar assay conditions, exponentially growing cultures of KB19,KB4 and KB25 were inoculated into AIF M2, pH 6.3, supplemented with 100mg/dL urea and with or without 100 μM NiCl₂ at initial cell density of10⁹ cfu/mL to determine whether the growth and rates of urea hydrolysisby these strains was dependent on the additional

Ni++. Urea concentration in the supernatants as a % of control andgrowth (OD600 nm) were measured every 4 hours. Four to nine independentexperiments were conducted and Student t-test was used for statisticalanalysis. Similarly, it was determined whether the growth and rate ofurea hydrolysis of these strains was dependent on urea concentration.Under similar growth conditions exponentially growing cultures of KB19,KB4 and KB25 were inoculated into AIF M2, pH 6.3, supplemented with 100μM NiCl₂ and 100, 200, or 300 mg/dL urea. Urea concentration in thesupernatants as a % of the control and growth (OD600 nm) were measuredevery 4 hours. Four to nine independent experiments were conducted andStudent t-test was used for statistical analysis. The survivability ofthese KB19, KB4 and KB25 was determined in artificial gastric juice inthe presence and absence of urea and dextrose. The average loss inviable cell count after exposure to artificial gastric juice (logscfu/mL) is shown in Table 2.

TABLE 2 PH/Additive KB19 KB4 KB25 1.4 7 7 7 2.0 7 7 7 2.5 3 4 4 2.5/Urea3 4 4 2.5/Dextrose 3 3 3 2.5/Urea + Dextrose 3 3 3 3.0 2 2 3 3.0/Urea 23 3 3.0/Dextrose 1 2 3 3.0/Urea + Dextrose 1 2 2 Initial cell densitywas 10⁷ cfu/mL. Urea and dextrose concentrations were 10 mg/mL and 1%,respectively.

Further it was determined whether the nutrient composition andavailablility had an affect on growth and urea hydrolysis by KB19, KB4,and KB25. Each strain was grown for 24 hours at 37° C. and pH 6.0 in thepresence or absence of 100 mg/dL of urea and combinations of nitrogenand carbon sources.

Further analysis of S. thermophilus KB19 indicated that this straincould survive a 3 hour exposure to gastric juice, pH 3.0, followed by a3 hour exposure to 0.3% oxgal, pH 6.0, with only 1 log loss inviability. Remaining viable cells were able to proliferate in AIF M2, pH6.0, supplemented with 230 mg/dL urea and completely hydrolyzed the ureawithin less than 18 hours (n=4). All test solutions were supplementedwith 230 mg/dL urea and 1% dextrose. Collectively, these analysesindicated that all three strains studied proliferated in the fed stateAIF medium in the pH range from 5.5 to 7.5, characteristic of colonenvironment; they could all use urea as a sole nitrogen source; and theyeach catabolized urea in the presence of other nitrogen sources. Ureahydrolysis was growth and pH dependent. Under the conditions tested, therate of urea hydrolysis was strain-dependent in tests of pH stability:KB19=KB25>KB4; Ni requirement: KB25>KB19>KB4; urea hydrolysis for over300 mg/dL: KB19=KB25>KB4; and specific nutrients: KB19>KB25>KB4.Further, there was strain-dependent results relating to survivability,wherein in tests of gastric juice stability: KB19>KB4>KB25; and bilestability: KB19>KB4>KB25.

In view of the desirable traits exhibited by S. thermophilus KB19 it wasfurther determined, using a disc-diffusion test, that K19 was sensitiveto Spectinomycin (100 μg), Kanamycin (30 μg), Chloramphenicol (30 μg),Spectinomycin (100 μg), Penicillin (10 IU), Carbenicillin (100 μg),Doxycycline (30 μg), and neomycin (30μg). To further enhance the levelsof urease secreted by strain K19, this strain was trained on increasinglevels of urea as described herein.

1. A protein-rich nutritional product comprising at least one probioticcomponent and at least one isolated edible protein.
 2. The nutritionalproduct of claim 1, wherein the probiotic component comprises aLactobacillus, Bacillus, Streptococcus Bifidobacteria, Saccharomyces orLeuconostoc.
 3. The nutritional product of claim 1, wherein the at leastone isolated protein comprises whey proteins or isolates, concentratesor hydrolysates thereof; milk proteins or isolates, concentrates orhydrolysates thereof whey growth factor extract; glutamine peptide; eggalbumen; soy proteins or isolates, concentrates or hydrolysates; orcaseinates.
 4. A method for removing nitrogenous waste products from theblood comprising administering to a subject in need thereof an effectiveamount of a nutritional product of claim 1 thereby removing nitrogenouswaste products from the blood of the subject.
 5. A method forameliorating renal failure comprising administering to a subject in needthereof an effective amount of the nutritional product of claim 1thereby ameliorating renal failure.
 6. The method of claim 5, whereinthe subject has a protein deficiency.